This proposal is focused on a fundamental question in genetics: How are chromosomes and the genes they carry appropriately segregated to the daughter cells at cell division? Defects in this process result in uneven partitioning of the genome that, in turn, causes aneuploidy. If this happens during gametogenesis, this type of genetic catastrophe results in spontaneous abortion or birth defects in resulting offspring. The gain or loss of an individual chromosome resulting from errors in mitotic cell division along with mutation of tumor suppressor genes or oncogenes contributes to early events in tumor formation and progression, and aneuploidy is a hallmark of almost all late stage tumors. The chromosomal element that controls its accurate segregation is the centromere. Molecular genetic and human patient data has defined centromere identity not by a particular DNA sequence, since centromeric ?-satellite repeats found at most normal centromeres are neither necessary nor sufficient to specify the location of a functioning centromere. Rather, the prevailing view is that centromere identity is defined epigenetically, and our long-term goal is to understand the basis for this epigenetic centromere mark and the mechanisms used for its establishment and maintenance. The leading candidate to represent the epigenetic mark is CENP-A, a histone H3 variant that assembles into nucleosomes at functional centromeres. Specific research in this proposal will address the following questions: How does CENP-A generate an epigenetic mark at the centromere? What impact does the incorporation of CENP-A into polynucleosome arrays have on their higher-order folding? Does the CENP-A-containing nucleosome directly recruit a specific set of centromere factors? Is there selective affinity of CENP-A for centromeric ?-satellite DNA? What is the mechanism for loading CENP-A onto centromeric DNA? What cellular proteins recognize CENP-A via its centromere targeting domain? Using a combination of biochemical reconstitution, biophysics, molecular genetics, and cell biology approaches we propose the following specific aims: 1. Determine the consequences of incorporation of CENP-A into centromeric nucleosomes 2. Define the requirements for CENP-A chromatin assembly at centromeres Together, these studies promise to provide valuable insight into the epigenetic mechanisms that specify and maintain the location of the centromere on the chromosome.